Designer interface peptide grafts target estrogen receptor alpha dimerization

Abstract

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide "I-box" derived from ER residues 503-518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479-485), LQQQHQRLAQ (residues 497-506), and LSHIRHMSNK (residues 511-520) and reported the suitability of using LQQQHQRLAQ (ER 497-506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents.

Keywords: Antiestrogen resistance; Breast cancer; Cell proliferation; Estrogen receptor; Interface peptides.

Fig. 1

Left panel: MD average structure of the four peptide in solution; Right panel: Evolution of secondary structure during simulation. A: Peptide I; B: Peptide II; C: Peptide III; D: Peptide IV.

Fig. 2

Variations of the RMSD and Rg during simulation time. A & C represents the RMSD and Rg profile all the four ERα-peptide complexes, respectively. B & D represents the RMSD and Rg profile all the four peptides when bound with ERα.

Fig. 3

Details of the van der Waals (A) and hydrogen bonding interactions (B) of the peptide I with ERα obtained from 10 ns MD simulation. Peptide I is colored blue while ERα is colored green. Interacting van der Waals residues are rendered as surface and colored red. Residues involved in the hydrogen bonding interactions are shows in stick representation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effect of ER interface peptides on MCF-7 breast cancer cell proliferation. A. Representative images from the Click-iT EdU-AlexaFluor 488 incorporation assay when MCF-7 breast cancer cells were treated with 1nM estradiol with or without ER interface peptide PI. The images were captured with a Zeiss Axio Observer inverted microscope with filters appropriate for Alexa Fluor 488- ex495nm, em519nm and NuclearMask (H10325) Blue stain- ex350nm, em461nm. The total DNA content was stained with a NuclearMask Blue stain (blue), and EdU was visualized after conjugation with Alexa Fluor 488 azide (green). Peptide treatment significantly reduced EdU incorporation (B). C. ER interface peptides (PI and PII) decrease estrogen induced cell proliferation of MCF-7 breast cancer cells in a High-throughput (HCS) Click-iT imaging assay. Fluorescence intensity was captured employing 497 mm excitation and 519 emission filters with the BioTek Synergy plate reader. Error analysis was performed using Graphpad Prism. Results represent mean ± standard deviation, where ** or †† represent p < 0.01. (* was use to identify statistical significance of treatment vs vehicle comparison and † comparisons between estrogen treatments). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)